The present invention relates to a method for producing dihydroxyacetone-3-phosphate using enzyme.
Dihydroxyacetone-3-phosphate is a substrate for fructose 1,6-bisphosphate aldolase and is used for stereoselectively synthesizing many kinds of carbohydrates by aldol condensation with various aldehydes. The carbohydrates thus synthesized can be widely utilized as medicines and their synthetic intermediates. The above reaction, however, requires large excesses of expensive dihydroxyacetone-3-phosphate, making the products expensive.
For example, dihydroxyacetone-3-phosphate can be chemically produced by directly phosphorylating dihydroxyacetone dimer with phosphorus oxychloride (POCl3) in pyridine (Tetrahedron Lett., 28, 1641 (1987)). This production method is complicated and thus cannot produce dihydroxyacetone-3-phosphate in a high yield at low cost.
Methods of producing dihydroxyacetone-3-phosphate using enzymes have also been reported. An example thereof is a method of synthesizing dihydroxyacetone-3-phosphate from dihydroxyacetone with immobilized glycerol kinase (GK) under conditions that ATP is regenerated by using phosphoenolpyruvic acid and pyruvate kinase (PK) (J. Org. Chem., 48, 3199(1983), and J. Am. Chem. Soc., 107, 7019(1985)). In this method, acetate kinase and acetylphosphate may be used instead of phosphoenolpyruvic acid and pyruvate kinase (J. Am. Chem. Soc., 107, 7019(1985)). These methods using glycerol kinase (GK) require highly purified enzymes and therefore cannot produce dihydroxyacetone-3-phosphate in a high yield at low cost.
For another example, it is theoretically possible to produce dihydroxyacetone-3-phosphate from dihydroxyacetone using dihydroxyacetone kinase (DHAK) (examined published Japanese patent applications (JP-B) No. Hei 4-29349 and Hei 4-22560). In particular, dihydroxyacetone kinase derived from yeast Schizosaccharomyces pombe is suitable for producing dihydroxyacetone-3-phosphate (JP-B Hei 4-29349). However, the cell extract of Schizosaccharomyces pombe contains coexisting enzymes such as phosphatases and triose-3-phosphate isomerase, which degrade the formed dihydroxyacetone-3-phosphate. The enzyme thus needs to be highly purified and has not yet been applied practically.
For the practical production of dihydroxyacetone-3-phosphate using enzymes, the enzyme or the biocatalyst containing the enzyme should have high reactivity, be prepared easily at low cost, and be highly purified so as not to degrade the product, dihydroxyacetone-3-phosphate. No efficient method for producing dihydroxyacetone-3-phosphate by using enzymes or biocatalysts containing the enzymes, which meets the above requirements, has been developed.
An objective of this invention is to provide a method, for efficiently producing dihydroxyacetone-3-phosphate.
Under the above circumstances, the present inventors have attempted to clone a gene encoding dihydroxyacetone kinase and to express it efficiently to obtain the practical enzyme. The present inventors have succeeded in overexpressing the enzyme in a microbial host. Furthermore, the present inventors have found that dihydroxyacetone-3-phosphate can be produced in a high concentration by contacting the host cells as they are or the enzyme produced by them with dihydroxyacetone, the substrate of the enzyme, thereby accomplishing the present invention.
More specifically, this present invention relates to:
(1) a method for producing dihydroxyacetone-3-phosphate, which comprises contacting dihydroxyacetone with bacterial cells transformed with the gene encoding dihydroxyacetone kinase or the enzyme produced by the bacterial cells;
(2) a method described in (1), wherein said dihydroxyacetone kinase is derived from yeast belonging to the genus Schizosaccharomyces;
(3) a method described in (1), wherein said dihydroxyacetone kinase is derived from Schizosaccharomyces pombe; 
(4) a method described in (1), wherein said bacterial cell is Escherichia coli; and
(5) a method described in any one of (1) to (4), wherein said contacting is performed under the conditions that coenzyme, ATP, is regenerated.
The source of the gene encoding dihydroxyacetone kinase used in the present invention is not limited. It is preferably derived from yeast belonging to the genus Schizosaccharomyces; more preferably, Schizosaccharomyces pombe; and still more preferably, Schizosaccharomyces pombe IFO 0354.The nucleotide sequence of the dihydroxyacetone kinase gene from Schizosaccharomyces pombe IFO 0354 is shown in SEQ ID NO: 1, and the deduced amino acid sequence is shown in SEQ ID NO: 2.
In the method of this invention, microorganisms transformed with dihydroxyacetone kinase capable of overexpressing the enzyme can be used as they are as an enzyme catalyst. The term xe2x80x9coverexpressionxe2x80x9d as used herein means a higher level expression than spontaneous |expression. In general, transformants capable of overexpresing the expression product can be obtained by introducing multiple copies of the expression unit with the gene encoding the expression product into host cells. The host bacterial cells used in the present invention are not particularly limited. It is preferable to use bacteria belonging to the genus Escherichia, and more preferable to use E. coli K-12 strain because various host-vector systems can be used. For example, pUC118 or pUC119 is the preferable expression vector into which the dihydroxyacetone kinase gene is to be inserted (Methods in Enzymology, 153, 3 (1987)). The gene can be introduced into the host cell by methods well known in the art such as a method described in xe2x80x9cMethods in Enzymology, 68, 299 (1979).xe2x80x9d For example, E. coli capable of overexpressing dihydroxyacetone kinase can be prepared as described below. First, the primers are designed for polymerase chain reaction (PCR) based on the database resulting from genome analysis of Schizosaccharomyces pombe. The target DNA is amplified by PCR using the primers and genomic DNA isolated from Schizosaccharomyces pombe IFO 0354 as a template DNA by conventional procedures. The PCR products are ligated into appropriate vectors and are cloned. The gene-inserted vectors are transformed into an appropriate strain of E. coli, and the transformants are then selected by detecting the existence of drug resistant gene(s) and the DNA insert(s). The DHAK productivity of the clones is measured by assaying the activity of the enzyme.
The enzyme produced by the bacterial cells can be isolated from the cells for use in the method of the present invention. The enzyme is not necessarily purified. A crude enzyme is used as well as the purified enzyme. The dihydroxyacetone kinase can be recovered from the grown cells by extracting the enzyme from the cells first because the enzyme is produced intracellularly. Namely, the cells are collected from the culture medium by filtration or centrifugation and disrupted by mechanical methods such as treatment with alumina, dynomill, or ultrasonication to extract the enzyme in a soluble form. Alternatively, the cell membranes are destroyed by treatment with organic solvents such as acetone, and the resulting cells are dried under reduced pressure. The powder thus prepared is used as a catalyst containing the enzyme. Insoluble materials are removed from the culture obtained in the methods described above by filtration or centrifugation to obtain the crude enzyme. Furthermore, the crude enzyme is concentrated and purified by methods known in the art such as adsorption chromatography, ion exchange chromatography, and gel filtration chromatography. The thus-obtained, partially purified preparation of dihydroxyacetone kinase is also used in the method of this invention.
The dihydroxyacetone kinase activity of the E. coli strain that overexpresses the enzyme or the enzyme isolated from said cells is measured by known methods (JP-B Hei 04-29349) as follows. The decrease of absorbance at 340 nm is spectrophotometrically measured during the reaction at 25xc2x0 C. in 1 ml of the reaction mixture containing 0.1M triethanolamine buffer (pH 7.5), 2.5 mM ATP, 4 mM MgSO4, 0.2 mM NADH, 2.5 units of glycerol-3-phosphate dehydrogenase (G3PDH), and 0.01 ml of a test enzyme solution (formula below).
One unit of the enzyme of the present invention is defined as the amount required to decrease 1 xcexcmole of NADH in 1 min under the conditions described above.
Dihydroxyacetone-3-phosphate is usually produced by the method of this invention under the following conditions. First, the reaction is carried out in a buffer with pH ranging from 7 to 8. Any buffer solution can be used as long as it can keep its pH within the above reaction pH range. Tris-hydrochloride buffer is preferable. The reaction temperature may be any range as long as dihydroxyacetone kinase used is active. Preferably, the temperature ranges from about 20xc2x0 C. to about 35xc2x0 C. It is necessary to add ATP to the reaction mixture since dihydroxyacetone kinase requires ATP as a coenzyme. However, the enzyme is inhibited by the formed ADP. Therefore, the reaction is preferably performed with regenerating ATP. Any known systems for regenerating ATP (J. Org. Chem. 48, 3199 (1983) and J. Am. Chem. Soc. 111, 627 (1989)) can be used. In view of the production cost, a system using acetyl phosphate and acetate kinase is preferred.
Dihydroxyacetone-3-phosphate produced by the above reaction can be identified by enzymatic and chemical techniques. Namely, the decrease of absorbance at 340 nm is spectrophotometrically measured during the reaction (formula below) at 30xc2x0 C. in 3 ml of a reaction mixture containing 0.1 M sodium acetate buffer (pH 6.0), 0.25 mM NADH, 0.6 units of glycerol-3-phosphate dehydrogenase (G3PDH), and 0.01 ml of a test solution. The concentration of dihydroxyacetone-3-phosphate (DHA phosphate) in the test solution is then determined based on a calibration curve which is made in advance using the standard compound.
After the above reaction, the reaction product can be precipitated by adding organic solvents such as ethanol to the reaction mixture, or by salting out, to recover it. Furthermore, the reaction product is purified by usual purification procedures such as column chromatography and recrystallization. Alternatively, it can be subjected to the next procedure as it is.